PROJECT SUMMARY In preterm neonates, excessive inflammatory responses have been implicated in necrotizing enterocolitis (NEC), the mechanisms for which are unclear. Microbiota and their metabolites including short chain fatty acids may modify the epigenome by DNA methylation and/or histone deacetylation, and specific microbiome patterns have been associated with altered DNA methylation of genes linked to lipid metabolism, obesity and inflammation. The long term goal of this research is to determine the mechanism for excessive inflammatory responses that lead to NEC and devise anti-inflammatory therapeutic and preventative strategies. The specific hypothesis of the proposed research is that epigenetic changes induced by the developing intestinal microbiome result in excessive immune and inflammatory responses that predispose to necrotizing enterocolitis in the preterm neonate. We intend to test this hypothesis by first establishing DNA methylation alterations in preterm infants with NEC compared to matched controls, in a nested cohort clinical design in Specific Aim 1. DNA methylation patterns will be delineated in intestinal epithelial cells and peripheral blood mononuclear cells and confirm differential gene expressions with transcriptomics in the intestinal epithelial cells from the stools and assess systemic and intestinal inflammation. In our second Specific Aim, we will test the hypothesis that specific microbiome and metabolomic signatures are associated with changes in the epigenome and transcriptome in preterm neonates with NEC. In the cohort enrolled, we will evaluate stool microbiome by metagenomics and stool and urine metabolome for microbial metabolites and record clinical metadata (e.g. gestational age, feeding) for multi-variable analysis. We anticipate distinct microbiome and metabolome signatures associated with DNA methylation alterations in NEC. Follow up studies of enteroids from surgical samples will be used to mechanistically test our hypothesis by measuring epigenetic changes after exposure to specific inflammatory, microbial or metabolomic stimuli that are identified from the proposed research. The proposed research is innovative as the epigenome alterations in relation to the microbiome and inflammation in the preterm infant have not been evaluated. We propose a holistic, multi-omics approach to delineate the mechanisms for excessive inflammation in the preterm infant. Our research will not only make an impact in the field of neonatology where major organ morbidity is related to inflammation but in also in other patients and diseases where inflammation is an inciting and key factor in the pathogenesis. Exploring the relationship between microbiota induced epigenetic changes and inflammatory responses may underpin the pathophysiology of NEC and lead to novel preventive strategies in the preterm neonate. Our research is significant because our work will provide new knowledge on microbiome-induced epigenetic changes that is relevant to a broad-spectrum of diseases such as cancer, infection and immunity.